Figure 3. PTENα is synthesized through an eIF2A-mediated CUG initiation mechanism and a palindrome sequence is essential for PTENα expression.

(A) Induction of PTENα by aurin tricarboxylic acid (ATA) in a time-dependent manner. HeLa cells were treated with ATA (80 mM) for various periods of time and the expression of PTENα as well as PTEN was examined by Western blot.

(B) Dose-dependent inhibition of PTENα expression by acriflavin. HeLa cells were treated with different doses of acriflavin for 4 h prior to immunoblotting for evaluation of PTENα expression. Expression levels of PTEN and GAPDH were included as controls.

(C) eIF2A alters the ratio of PTENα versus PTEN by up-regulating PTENα and down-regulating PTEN. FLAG-tagged eIF2A was overexpressed in HEK293T cells prior to Western evaluation of PTENα and PTEN. eIF2A expression was verified by probing the same blot with anti-FLAG antibody. GAPDH was used as a loading control.

(D) Reduction of PTENα in response to knockdown of eIF2A. HeLa cells were infected with lentivirus expressing shRNA of eIF2A or scramble shRNA. Cell lysates were analyzed by Western blotting with antibodies against eIF2A, PTEN (m) and GAPDH.

(E) CTG⁵¹³-centered palindromic sequence and mutagenesis disruption of the palindrome.

(F) Abolition of PTENα by palindrome disruption. Mutations were made at CTC⁵¹⁰, thetriplet immediately before the CTG⁵¹³ start codon, or at CUG⁵¹³ itself as indicated prior to Western analysis of PTENα expression.

See also Figure S3.